an optimized, broadly applicable piggybac transposon...

Nucleic Acids Research, 2017 1–13doi: 10.1093/nar/gkw1290

An optimized, broadly applicable piggyBactransposon induction systemZongtai Qi, Michael Nathaniel Wilkinson, Xuhua Chen, Sumithra Sankararaman,David Mayhew and Robi David Mitra*

Department of Genetics and Center for Genome Sciences and Systems Biology, Washington University, School ofMedicine, St. Louis, MO 63108, USA

Received April 11, 2016; Revised November 01, 2016; Editorial Decision December 10, 2016; Accepted January 1, 2017

ABSTRACT

The piggyBac (PB) transposon has been used in anumber of biological applications. The insertion ofPB transposons into the genome can disrupt genesor regulatory regions, impacting cellular function, sofor many experiments it is important that PB trans-position is tightly controlled. Here, we systematicallycharacterize three methods for the post-translationalcontrol of the PB transposon in four cell lines. We in-vestigated fusions of the PB transposase with ERT2and two degradation domains (FKBP-DD, DHFR-DD),in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition ef-ficiency of the activated construct and (iii) the ef-fects of two inducer molecules on cellular transcrip-tion and function. We found that the FKBP-DD con-fers the PB transposase with a higher transposi-tion activity and better dynamic range than can beachieved with the other systems. In addition, wefound that the FKBP-DD regulates transposon activ-ity in a reversible and dose-dependent manner. Fi-nally, we showed that Shld1, the chemical inducerof FKBP-DD, does not interfere with stem cell differ-entiation, whereas tamoxifen has significant effects.We believe the FKBP-based PB transposon inductionwill be useful for transposon-mediated genome en-gineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.

INTRODUCTION

Transposons are genetic elements that are able to mobilizethroughout a host genome autonomously. Due to their mo-bility, DNA transposons and retrotransposons have beenwidely used as tools for generating mutation libraries andfor delivering non-viral gene constructs into the genomesof a variety of organisms (1). The use of DNA transposons

in mammalian genomes, however, was initially impeded bya lack of active transposable elements. The synthetic resur-rection of Sleeping Beauty, a Tc1/mariner-like transposableelement of the salmanoid fish genome, initiated the develop-ment of transposon technologies for use in mammalian cells(2). Since then, several other transposon systems have beendeveloped (3–5), including the piggyBac (PB) system, de-rived from the cabbage looper moth Trichoplusia ni (6). Thissystem is widely used because the PB transposon consis-tently exhibits high transposition efficiencies in different celllines and organisms (7), can transpose cargos of up to 100kb without a significant reduction in efficiency (8), mobi-lizes without leaving footprint mutations at the excision site,and is amenable to molecular engineering (9). In addition,the PB system is highly efficient for germ line insertionalmutagenesis in mice and lacks overproduction inhibition(10). These unique characteristics make the piggyBac trans-poson an invaluable tool for a wide range of applications,including stable gene delivery (11), transgene excision (12),insertional mutagenesis (13) and the mapping of transcrip-tion factor (TF) binding in eukaryotic genomes (14–16). Formany of these applications, the PB transposon is engineeredto act as a gene trap that will disrupt genes when insertedinto the genome (13), so it is useful to be able to tightly con-trol PB transposition so that insertion only occurs duringthe proper experimental window, and not, for example, dur-ing the propagation of cell lines or mice. For other applica-tions, such as the mapping of TF binding to genomic DNAby PB transposition (14–16), the constitutive activity of thePBase prevents the determination of the precise timing ofthe binding events, a feature that is particularly useful whenstudying developmental processes. Therefore, a method forinduction that can tightly regulate the temporal activity ofthe PB transposon system is desirable. Furthermore, post-translational control is appealing because protein activitycan be switched on or off considerably faster than is possi-ble with transcriptional control schemes (17,18).

Prior to the work presented here, the only exist-ing inducible PB transposon system that operated post-

*To whom correspondence should be addressed. Tel: +1 314 362 2751; Email: [email protected] Address: Robi David Mitra, Room 4301, Washington University School of Medicine, 4515 McKinley Ave, St. Louis, MO 63108, USA.

C⃝ The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), whichpermits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please [email protected]

2 Nucleic Acids Research, 2017

translationally utilized the mutated ligand-binding domainof the estrogen receptor (ERT2) (9). The PBase-ERT2 fu-sion is constitutively expressed but is sequestered outsideof the nucleus by the cytoplasmic heat shock protein 90(HSP90), which binds the ERT2 domain (Figure 1A). In thepresence of a small molecule inducer (4-Hydroxy tamox-ifen; 4OHT), HSP90 binding is abolished and the PBase-ERT2 fusion rapidly relocates to the nucleus where it di-rects transposition. The PBase-ERT2 fusion works well inseveral contexts (14), but our early experiences with this sys-tem revealed that dynamic range of induction varies widelybetween different cell types, possibly due to differences inHSP90 levels, which is not constant across cell types (19–22). We further found that ERT2-PBase fusions are nothighly active when induced (relative to the unfused trans-posase, see results). Finally, we found that the chemical in-ducer 4OHT inhibited the in vitro differentiation of mouseembryonic stem cells, an observation consistent with simi-lar reports that 4OHT adversely affects neurogenesis (23),myelinogenesis (23), myometrial differentiation (24) andsexual maturation (25).

In light of these observations, we sought to develop a PBtransposon induction system that provides a tightly regu-lated transposase enzyme that displays a large differencein activity between the induced and un-induced state, that,when induced, deposits transposons with an efficiency equalto that of the native protein, that is highly active across awide variety of cell types, and that is induced with a smallmolecule that has minimal effects on general cellular func-tions. To do so, we characterized and optimized fusionsof the PB transposase with two different destabilized do-main (DD) proteins, FK506- and rapamycin binding pro-tein (FKBP) (17) and dihydrofolate reductase (DHFR) (18),and compared these to the ERT2 based induction system (9)in four different cell lines. Destabilized domains are small,inherently unstable proteins that bind small molecules andhave been destabilized by mutation. When fused to PBase,the mutated domain unfolds and the fusion protein is de-graded by the proteosome (Figure 1B). However, if the cog-nate small molecule ligand is provided, the DD is stabi-lized and the PBase fusion protein is rescued from degra-dation and transposase activity is restored in a rapid, dose-dependent and reversible manner. Unlike the ERT2-basedsystem where induction is mediated by a specific cytoplas-mic protein HSP90, DD degradation is mediated by theubiquitin–proteasome pathway, a common protein degra-dation mechanism that is active in all mammalian cells(26,27).

To identify the optimal configuration for each of the threeinduction systems, we investigated 15 different fusions ofthe PB transposase with FKBP, DHFR or ERT and mea-sured the fold-induction of activity between the uninducedand induced states, the maximum transposition rate of theactivated constructs as compared to the unfused piggyBactransposase, and the effects of two of the inducer moleculeson cellular transcription and differentiation.

MATERIALS AND METHODS

Plasmid construction

The plasmids that contain mutated destabilized domains,FKBP and DHFR, were purchased from Addgene with ID31763 and 29326, respectively. The PBase-ERT2 plasmid(mPBase-L3-ERT2) was a kind gift from Dr Bradley (9).The coding sequence of mPBase-L3-ERT2 was sub-clonedinto a yeast shuttle vector pRS314 containing CEN6, ARSand TRP1 to use ‘gap repair’ cloning technique in yeastcells (Strathern and Higgins 1991). This engineered yeastshuttle vector was used as a ‘parental’ plasmid (pRM1056)to derive other variants of PBase constructs by gap repairmethod (15). Briefly, PCR-generated sequences were clonedinto linearized vectors by recognizing a 40 bp overlap attheir ends. This 40 bp overlap can be engineered by design-ing primers for amplification of the desired sequences. Forexample, to replace the ERT2 sequence with the FKBP se-quence, the pRM1056 plasmid was linearized by restrictiondigestion that cut the plasmid within the ERT2 sequence.The FKBP sequence was amplified with a pair of primersthat have a 40 bp sequence that is homologous to pRM1056.The FKBP PCR products and linearized pBM1056 were co-transformed into yeast cells and the yeast cells were selectedfor Trp+ colonies. DNA extracted from Trp+ yeast colonieswas introduced into E. coli. Finally, the plasmid was iso-lated by QIAprep Spin Miniprep Kit (QIAGEN) and wasconfirmed by Sanger sequencing. The engineered constructsused in this study are depicted in Figure 2.

Cell culture and neural differentiation

Human embryonic kidneys cell lines (HEK293 andHEK293T) and human colon adenocarcinoma cell lineHCT116 were maintained in Dubecco’s Modified EagleMedia (DMEM; Gibco) supplemented with 10% fetalbovine serum. RW4 mouse embryonic stem cells (ESCs)were cultured in complete media consisting of DMEM sup-plemented with 10% new born calf serum, 10% fetal bovineserum (Gibco) and 0.3 mM of each of the following nucle-osides: adenosine, guanosine, cytosine, thymidine and uri-dine (Sigma-Aldrich). To maintain their undifferentiatedstate, cells were also cultured in the presence of 1000 U/mlleukemia inhibitory factor (LIF; Chemicon) and 20 mM !-mercaptoethanol (BME; Invitrogen) in flasks coated with a0.1% gelatin solution (Sigma-Aldrich).

Mouse ESCs were differentiated into ventral spinal neu-ral cells using a retinoic acid (RA) and smoothened agonistinduction protocol as described (28). Briefly, ESCs were cul-tured in suspension on low attachment plates (Corning) inmodified DFK5 media consisting of DMEM/F12 base me-dia (Gibco) containing 5% knockout serum replacement, 1xinsulin transferrin selenium, 50 "M of non-essential aminoacids, 100 mM of BME, 5 mM of thymidine and 15 mMof the following nucleosides: adenosine, cytosine, guanosineand uridine. During this process, ESCs aggregate into multi-cellular embryoid bodies (EBs). After the first 2 days, theEBs were moved to a 15 ml of conical and allowed to set-tle for 5 min. The media was aspirated and replaced with10 ml of fresh DFK5 containing 2 mM of RA and 600 nMof smoothened agonist (Millipore). EBs were then cultured

Nucleic Acids Research, 2017 3

Figure 1. Schematic illustration of the ERT2 and destabilized domain (DD) based PiggyBac (PB) transposon induction systems. (A) For the ERT2-basedPB transposon induction system, the PBase-ERT2 fusion is constitutively expressed but sequestered outside of the nucleus by HSP90, which binds theERT2 domain. In the presence of ER antagonist 4-OHT, HSP90 dissociates and the PBase-ERT2 fusion rapidly relocates to the nucleus where it directstransposition. (B) For the DD-based PB transposon induction system, a DD (either FKBP or DHFR) was fused to the PBase. The DD confers theinstability to the fused protein such that the PBase fusion protein was constitutively degraded. However, binding of a small molecule ligand (Shld1 forFKBP; TMP for DHFR) to the DD prevents PBase from degradation and stabilizes it.

Figure 2. Schematic illustration of the constructs used in this study. For the helper constructs: PBase, PiggyBac transposase; CMV, cytomegaloviruspromoter; pA, bovine growth hormone polyA signal. FKBP, a destabilizing domain derived from FK506 and rapamycin binding protein. DHFR, adestabilizing domain derived from Escherichia coli dihydrofolate reductase; YFP, yellow fluorescent protein; ERT2, mutated ligand-binding domain of theestrogen receptor; Cumate, cumate operator (mutated cmv promoter). For the donor construct: 5′TR-puro-3′TR contains the minimal PB terminal repeats(5′LR and 3′TR) flanking a PGK promoter driven puromycin resistance cassette; EF1, elongation factor 1 promoter; GFP, green fluorescent protein.

on the adhesive plates (Corning) for an additional 4 daysfor further differentiation, and media was replaced every 2days.

Cell transduction and transgenic cell line generation

All lentiviruses used for cell transduction were produced bythe Hope Center Viral Vectors Core at Washington Univer-

sity School of Medicine. A CymR-expressing HEK293 cellline was made by transducing the cell with lentivirus con-taining the cumate operator repressor driven by an EF1apromoter at a multiplicity of infectivity of 20 to ensurethat CymR is overexpressed. Two days after transduction,neomycin was added to the culture medium (500 ng/ml)and maintained for 7 days. Then, the CymR-expressing


HEK293 cell line was further transduced with lentiviruscontaining an FKBP-PBase-FKBP-IRES-RFP fusion genedriven by a cumate inducible promoter at multiplicity of in-fectivity of 0.5 to favor single-copy integration. To obtain apure population, transduced cells were then sorted for redfluorescent protein (RFP) positive cells.

Cell transfection and drug administration

All plasmids used for the transfection of cells were preparedusing EndoFree Plasmid Maxi Kit (Qiagen) following themanufacturer’s protocol. About 105 cells were electropo-rated with a total of 0.6 "g of DNA (0.1 "g helper plasmidand 0.5 "g donor plasmid) by the Neon transfection system(Invitrogen) and plated to one well of a 6-well plate. Im-mediately upon transfection, cells were treated with 1 "M4-hydroxy tamoxifen (4OHT, Sigma) for the ERT2 basedinduction system, 1 "M Shld1 (Clontech) for FKBP basedinduction system, 10 "M trimethoprim (TMP, Sigma) forthe DHFR based induction system. Negative controls weremock-treated with 95% ethanol as vehicle. For the trans-genic cell line expressing inducible PBase driven by a cu-mate promoter, only donor plasmid was used in transfec-tion. Cumate inducer (System Biosciences) was added atconcentrations ranging from 0 "g/ml to 70 "g/ml to reg-ulate the strength of cumate promoter. For the purposeof reproducibility, experiments were done in triplicates. Toselect cells in which the PB transposon transposed, cellswere trypsinized 2 days after transfection and cultured on10 cm dish (Corning) with 10 ml fresh media containingpuromycin (1 "g/ml). The puromycin selection normallytakes 7 days before visible cell colonies are formed.

For experiments testing the reversibility of the FKBP-based PB transposon system, Shld1 (1 "M) was added tothe culture medium immediately after transfection. One dayafter transfection, a small aliquot of cells were used forimaging and flow cytometry analyses and the rest of thecells were passaged to a 12-well plate and provided freshShld1-free medium. The passaged cells were grown to about100% confluence before another passage to a 12-well plate.One well of cells were used for imaging and flow cytometryanalyses every day. After 3 days of destabilization, the cellswere treated with fresh medium containing 1 "M Shld1 tore-stabilize the FKBP-YFP-PBase fusion protein. The re-stabilization lasted for another 3 days during which the cellswere analyzed by flow cytometry every day.

Cell colony staining and counting

The visible drug-resistant cell colonies were fixed with phos-phate buffered saline (PBS) containing 4% paraformalde-hyde for 1 h and then stained with 1% methylene blue in 70%EtOH for 30 min, washed in distilled water and air-driedovernight. Colonies with diameters more than 0.3 mm werecounted by ImageJ software (National Institutes of Health).The number of puromycin-resistant colonies formed fromthe cells transfected with both donor and helper plasmidswas normalized to that with donor and helper backboneplasmids (yeast shuttle vector pRS314) prior to any fur-ther calculations. The transposition activity of a PBase fu-sion protein was calculated as the normalized number of

colonies from the inducible domain (i.e. FKBP, DHFR orERT2) fused PBase divided by that from ‘wild type’ unfusedPBase. For experiments that used the transgenic cell lineexpressing FKBP-PBase-FKBP driven by a cumate pro-moter, we used normalized number of puromycin-resistantcolonies to estimate the transposition activity due to the un-availability of the cell line expressing ‘wild-type’ unfusedPBase. Fold-induction was calculated as the normalizednumber of colonies from the induced samples divided bythat from untreated samples.

Imaging and flow cytometry

Fluorescent images were taken on Zeiss Axioskop fluores-cence microscope equipped with a QICAM FAST 1394digital CCD camera. Cells were grown to 90% confluence,trypsinized from the plate and suspended in PBS, washedonce with PBS and resuspended in Hank’s balanced salt so-lution supplemented with 2 mM ethylenediaminetetraaceticacid. Cellular fluorescence was analyzed on an iCyt Reflec-tion HAPS2 cell sorter at the Washington University Site-man Flow Cytometry Core. The gate was set relative to thecells transfected with non-fluorescent control plasmids toeliminate background. Cells transfected with a positive con-trol fluorescent reporter plasmid were also used to elimi-nate false positive singles. About 10 000 cells were analyzedfrom each FACS and post-sort analysis was performed withFloJo software to obtain the percentage of fluorescent pos-itive cells.

RNA extraction and sequencing

Total RNA was isolated from the RW4 mouse ESCs us-ing the PureLink RNA Mini kit (Ambion) according tothe manufacturer’s instructions. The quantity of RNA wasmeasured using a spectrophotometer (NanoDrop 2000c;Thermo Scientific). Samples with a RNA concentration(A260/A280 ≥1.8 ng/"l) and purity (A230/A260 ≥ 2.0ng/"l) were selected. The Agilent 2100 Bioanalyser wasused to determine the RNA integrity number. The degra-dation level was identified using the RNA 6000 NanoLabChip kit (Agilent). Samples with RNA integrity number> 9.8 were further processed using TruSeq mRNA LibraryPreparation Kit (Illumina) and then sequenced by IlluminaMiSeq platform at the Genome Technology Access Centerat Washington University in St. Louis. The gene expressiondata generated for this study can be found under the NCBIGene Expression Omnibus accession number GSE78857.The expression data is also publicly available at the Centerfor Genome Sciences by request.

Reads mapping and statistical analysis

Trimmomatic (v0.32) (29) was employed on RNA-SeqFASTQ files to clip the illumina adaptors and remove thereads of low quality. The cleaned reads were mapped backto mm10 genome reference from UCSC database usingSTAR (v2) (30). We used the HTSeq package (31) to esti-mate the count of uniquely mapped reads for each of the an-notated genes in the mm10 gene transfer format (.GTF) file.Differential expressed genes were analyzed with R (v 2.13.0)


using DESeq (v1.4.1) (32) available in Bioconductor (v 2.8).The resulting P-values were adjusted using the Benjamini–Hochberg correction and only genes that were significant ata false discovery rate of 0.05 were considered as expressed.For other comparisons between different experimental con-ditions, the statistical significance was assessed by pairedStudent’s t-test and a P-value < 0.05 was considered sig-nificant.

RESULTS

Overview

We evaluated and optimized three induction systems fortheir abilities to provide tight post-translation control overthe PB transposon. We used one degradation domain (DD)derived from human FKBP12 (FKBP) (17) and anotherfrom E. coli DHFR (18) and compared these against theERT2-based system in four different cell lines: human em-bryonic kidney cells (HEK293 and HEK293T), human col-orectal cancer cells (HCT116) and RW4 mouse ESCs. Toobtain the most efficient induction possible, a number ofdifferent fusions constructs were investigated for each sys-tem (Figure 2). We fused DD or ERT2 induction domainsin-frame at the N terminus, C terminus or at both terminiof the PBase. For N terminal fusion proteins, an 18 aminoacid linker sequence was included between the PBase andthe induction domain (33). For the C terminal fusion pro-tein, a 24 amino acid linker was added between the PBaseand induction domain (9).

In order to evaluate these different fusion proteins, weneeded a simple, robust way to measure PB transposition.We adopted the commonly used method of co-transfectingcells with two plasmids: a ‘helper’ plasmid expressing thePBase and a ‘donor’ plasmid carrying the PB transposonwith a drug-resistance marker (15). Once transfected intothe host cells, the PBase mobilizes the transposon from thedonor plasmid and integrates it into the host genome, con-ferring the cell with drug resistance. The number of drug-resistant colonies is closely correlated with the number oftransposon integration events and is a key indicator of theefficiency of the PB transposon system (9,15,34). We usedthis system to measure, for each post-translational controlsystem, the absolute transposition efficiency of the activatedconstruct relative to the unfused piggyBac transposase, andthe fold-induction achieved by each method in four differentcell lines. We then went on to optimize the most promisingsystem, characterize the dynamics of the temporal controlachieved by the optimal system and evaluate the effects oftwo of the inducer molecules on cellular transcription andfunction.

Evaluating the transposition efficiencies of inducible PBase

We first measured the transposition activity of the inducedPBase fusion proteins relative to that of unfused PBase.The transposition activity for each variant of helper con-structs was obtained by transfecting the above-mentionedfour cell lines with an equimolar ratio of donor and helperplasmids in the presence of chemical inducers (Shld1 forFKBP fusions; TMP for DHFR fusions; 4OHT for ERT2fusions). Typical images of stained colonies from negative

control, inducer-treated and non-treated co-transfected ex-periments, and positive control with unfused PBase areshown in Figure 3A. The induced transposition activitiesrelative to unfused PBase are shown in Figure 3B, and rawcolony counts are provide in Supplementary Figures S1 andS2. In our assays, the DD-based induction systems hadsignificantly higher activities than the ERT2-based induc-tion system in all cell lines. The highest PBase activity wasobserved for the constructs of FKBP-PBase and DHFR-PBase, both of which had activities that were ∼95% of‘wild-type’ levels. In contrast, all of the ERT2 fusion pro-teins displayed activities less than 25% that of the unfusedPBase. Thus, we conclude that, under inducing conditions,the FKBP and DHFR DDs have little effect on PBase trans-position activity while ERT2 appears to significantly inter-fere with the PBase activity. For the two DD-based induc-tion systems, the transposition activities between N and Cterminally tagged PBase fusions are not significantly differ-ent (P > 0.05). However, we observed a significant decreaseof PBase activity when both termini were attached to eitherDD, suggesting that at least one terminus should be exposedfor near-optimal PBase transposition activity.

The FKBP DD system achieved the highest fold-change be-tween induced and uninduced states

We next sought to quantify how tightly the different induc-tion systems regulated the PBase protein. To do so, we againtransfected the four cell lines with donor and helper plas-mids; however, in this assay, we omitted the small-moleculeinducers. We found that the DHFR-based PB induction sys-tem had a much higher background than did the FKBP andERT2-based ones (Figure 3C). To determine which PBasefusion protein provided the largest dynamic range, we usedthe corresponding induced and non-induced transpositionrates to calculate the fold-induction for each protein (Figure3D). This analysis revealed that the FKBP-based inductionsystem displayed a significantly higher fold-induction thanboth the DHFR and ERT2-based systems in all cell lines(P < 0.05). The FKBP-PBase-FKBP fusion protein showedthe highest fold-induction while the ERT2-PBase-ERT2 fu-sion protein showed the lowest. Taking fold-induction andthe absolute activity of induced PBase into consideration,the FKBP-based inducible PB transposon system outper-formed its DHFR and ERT2-based counterparts.

HSP90 levels correlate with the dynamic range of the ERT2system

The dynamic ranges of the ERT2 controlled PBase fu-sion proteins were highly variable across the different celllines (Figure 3D). For example, the PBase-ERT2 fusion dis-played a 17-fold change in transposase activity between in-duced and uninduced conditions in mouse RW4 embry-onic stem cell, but a significantly lower fold-induction inthe other cell lines (P < 0.05). One possible explanationfor this observation is that the HSP90 protein is typicallyhighly expressed in mouse ES cells. HSP90 is the moleculethat sequesters the fusion protein in the absence of inducer,and it has been previously reported the HSP90 RNA (35)and protein (36) levels are significantly higher in mouse ES


Figure 3. The performance of different helper constructs in DD and ERT2 based PB transposon induction systems. (A) Typical images of colony formingand staining assays to evaluate the transposition efficiency. The scale bar in the enlarged image equals 0.1 mM. The construct used is FKBP-PBase. (a)Cells transfected with donor and helper backbone plasmids (yeast shuttle vector pRS314) were used to estimate the background or random insertions. (band c) Cells transfected with FKBP-PBase and donor plasmids either in the absence or presence of Shld1 were used to evaluate the transposition efficiency.(d) Cells transfected with unfused PBase and donor plasmid were used to estimate the maximum transposition efficiency. (B) Induced PBase activity ofdifferent PBase fusions relative to wild-type PBase across four cell lines. The number of puromycin-resistant colonies formed from the cells transfectedwith both donor and helper plasmids was normalized to that with donor and helper backbone plasmids prior to any further calculations (backgroundsubtraction). The induced transposition activity of an inducible domain (i.e. FKBP, DHFR or ERT2) fused PBase was calculated as the normalized numberof colonies from the PBase fusion divided by that from ‘wild type’ unfused PBase. Experiments were done in triplicate. (C) Non-induced PBase activity ofdifferent PBase fusions across four cell lines. Experimental conditions were the same as in B except that no drug was added for the non-induced samples.(D) Fold induction of different PBase fusions across four cell lines. The induction fold was calculated as the normalized number of colonies from inducedsamples in B divided by that from untreated samples in C.


cells than in differentiated cell lines or embryoid bodies. Toconfirm HSP90 is indeed highly expressed in our mESCs,we performed RNA sequencing on the RW4 and HCT116lines. We found that the beta-actin normalized HSP90 ex-pression level in the RW4 mouse ES cell line was about4-fold higher than that in HCT116 cell line (Supplemen-tary Data 1 and 2), which is consistent with the theory thatHSP90 levels explain the differences observed between RW4mouse ESCs and HCT116 cells.

There is a trade-off between maximal PBase activity and fold-induction

In the FKBP-based induction system, the PBase fusionprotein shuttles between the cytoplasm where FKBP-directed degradation occurs and nucleus where PB transpo-sition occurs. Therefore, we hypothesized that this nucleo-cytoplasmic transport could be fine-tuned to create an in-duction system with both efficient transposition and rapiddegradation. To try to further improve transposition effi-ciency, we fused the nuclear localization sequence (NLS)from the SV40 large T-antigen upstream of, and in-framewith, the FKBP-PBase fusion, which had the highest trans-position activity in our previous tests (Figure 3B). We rea-soned that the NLS should direct more fusion protein tothe nucleus, increasing the transposition rate upon admin-istration of Shld1. We used transient transfection to in-troduce an equimolar ratio of NLS-FKBP-PBase fusion-carrying plasmid and donor plasmid across four differentcell lines, and compared the results to that of FKBP-PBaseplasmid without the NLS. After induction, the engineeredfusion protein (NLS-FKBP-PBase) displayed an increasein transposition activity relative to the FKBP-PBase fusionprotein (Figure 4A, P < 0.05). However, we also observedhigher background transposition in the uninduced samplefor this construct. As a result, the fold-induction for theNLS-FKBP-PBase fusion was lower than that of FKBP-PBase (Figure 4B, P < 0.05). This suggests that the NLS se-quence sequesters some protein in the nucleus and preventsefficient degradation in the absence of Shld1. Based on thisobservation, we concluded that adding NLS to PBase fu-sion protein is not optimal for an inducible PB transposonsystem.

To test the possibility of further reducing the backgroundtransposition that occurs in the absence of chemical inducer,we made two more fusion constructs. One contains two tan-dem FKBP domains at the N terminus of PBase (Figure 2,construct 5) since our previous results showed that an N-terminally tagged PBase was more tightly regulated thanthe corresponding C-terminally tagged PBase. The secondconstruct utilized three FKBP domains: two in tandem onthe N-terminus and one on the C-terminus (Figure 2, con-struct 6). We tested the transposition efficiency and dynamicrange of these constructs as before, and found that thetriple FKBPs fusion protein (FKBP-FKBP-PBase-FKBP)showed the highest fold-induction in all cell lines (Figure4A, right panel), followed by the double FKBPs fusion pro-teins (FKBP-PBase-FKBP and FKBP-FKBP-PBase). Allthree constructs achieved a significantly higher fold induc-tion than did FKBP-PBase (P < 0.05).

We next examined the maximal induced transposition ef-ficiency of these constructs relative to the unfused PBase(Figure 4B, right panel). We found that the transpositionefficiencies of FKBP-FKBP-PBase-FKBP, FKBP-FKBP-PBase and FKBP-PBase-FKBP were 60%, 79% and 80%of the unfused PBase, which is significantly lower than the95% relative activity we observed for FKBP-PBase (P <0.05), indicating that transposition activity gradually de-creased when more FKBP domains were added to PBase.Taken together, these results suggest that there is a trade-offbetween the efficient degradation of the PBase fusions un-der non-inducing conditions and the enzymatic activity ofthese fusions under fully inducing conditions (Figure 4C).Specifically, if high activity upon induction is the objective,then the FKBP-PBase fusion is optimal, while if a high fold-change in induction is required, the FKBP-FKBP-PBase-FKBP fusion is the best choice.

Fusion protein levels can be tuned to achieve low backgroundtransposition and high inducibility

For some applications, it is not important to obtain themaximum possible PBase activity, but it is instead prefer-able to have an assay with low background levels and toachieve a large fold-change in activity upon induction. Wereasoned that the high background levels observed in Fig-ures 3 and 4 might be due to the fact that the PBase-DDfusion proteins were all highly expressed and therefore theproteasome was getting overloaded. To test this hypothesis,we created lentivirus containing an FKBP-PBase-FKBP-IRES-RFP fusion gene under the control of a cumate in-ducible promoter (37) (Figure 2, construct 9), which allowsgene expression to be tuned by varying the cumate concen-tration in the culture media. Next, we infected cymR ex-pressing HEK 293 cells, and isolated transduced cells byperforming FACS to purify RFP positive cells. We trans-fected these cells with donor plasmid (Figure 2, construct16), titrated FKBP-PBase-FKBP protein levels by culturingthe cell with varying amounts of cumate, in the presence orabsence of Shld1, and determined PBase activity by count-ing puromycin resistant colonies as previously described.The results are shown in Figure 5. As lower concentra-tions of cumate were added to the media, much lower back-ground and larger fold-changes in induction were achieved.For example, when 10 "g/ml cumate was added to the me-dia, no background transposition was observed, and we ob-served a more than 100-fold change in PBase activity afterinduction with Shld1. These results demonstrate that, whenthe FKBP-PBase-FKBP protein is expressed at moderatelevels, this system has essentially no background transposi-tion yet is still robustly inducible. This level of control comesat some cost, however, as the maximum level of PBase ac-tivity is roughly one-fourth the maximum rate we observed(i.e. compare the 10 "g/ml cumate and Shld1 culture condi-tion to the PBase activity of cells grown with 70 "g/ml cu-mate and Shld1). For experiments that do not require rapid,post-transcriptional induction, it is possible to have the bestof both worlds, namely zero background and high absoluteactivity, by growing cells in the absence of cumate and Shld1and then adding both chemicals to induce transposition.


Figure 4. Optimization of the induced transposition activity and fold induction for FKBP-based PB transposon induction system. (A) Induced trans-position activity of different PBase fusion proteins relative to wild-type PBase across four cell lines. Experiments were done in triplicates. The inducedtransposition activity of a PBase fusion protein was calculated as the normalized number of colonies from the PBase fusion in the presence of corre-sponding chemical inducer divided by that from ‘wild type’ unfused PBase. (B) Fold induction of different PBase fusion proteins across four cell lines. Theinduction fold was calculated as the normalized number of colonies from chemical inducer treated samples divided by that from untreated samples. (C)Comparison of the induced transposition activity with the fold induction for different PBase fusion proteins across four cell lines.


Figure 5. The performance of the cumate-regulated FKBP-based PB transposon induction system. Cumate was included in culture media at concentrationsranging from 70 "g/ml (full induction of the cumate promotor) to 0 "g/ml (no induction). For each cumate concentration, Shld1 (yellow) or vehicle (70%ethanol, blue) was added to the medium to activate the PB transposon induction system. The normalized number of puromycin-resistant colonies and thefold-activation are plotted at different cumate/Shld-1 concentrations.

The FKBP-PBase-FKBP protein levels can be tuned post-transcriptionally

We next sought to determine if PB activity could be post-transcriptionally tuned by adjusting Shld1 concentration.To do so, we transfected four cell lines with donor andhelper plasmids (Figure 2, construct 3 and 16) and subjectedthese transfected cells to various concentrations of Shld1: 0nM, 8 nM, 40 nM, 200 nM and 1 "M. We calculated thenormalized number of puromycin resistant cell colonies foreach Shld1 concentration. We found that the colony numberincreased almost linearly with the increase of the concentra-tion across all cell lines (r = 0.81, Figure 6A), indicating theFKBP-based PB transposon induction is tunable and dose-dependent.

Counting puromycin-resistant colonies reveals the num-ber of cells with at least one integrated transposon; however,it does not provide information about the average numberof transposition events that have occurred per cell. To ad-dress this, we also measured PBase activity at various con-centrations of Shld1 using an alternative readout for trans-position. We created a donor vector (Figure 2, construct 17)in which a GFP gene is split by a PB transposon, render-ing it inactive (Supplementary Figure S3A). Nuclear PBasewill excise the transposon creating a functional GFP genethat is then expressed, and so the average fluorescence ofthe cell population is proportional to the number of trans-poson excision events. The results of these experiments areshown in Supplementary Figure S3B and S3C. The meancellular fluorescence increases monotonically with Shld1 ina near-linear fashion, validating the results obtained withthe puromycin donor, and supporting the thesis that PB in-duction is tunable and dose-dependent.

The FKBP based PBase system is reversible

To determine if the degradation of FKBP-PBase is re-versible, we fused a yellow fluorescent protein (YFP) in-

frame between FKBP and PBase by an 18 amino acid linkerat N terminus of PBase (33) (Figure 2, construct 7), al-lowing the visualization and semi-quantitative analysis ofthe expression of the PBase fusion by monitoring fluores-cence intensity. The four cell lines mentioned above weretransfected with the helper plasmid (FKBP-YFP-PBase)and separately, a control plasmid (YFP-PBase). Shld1 wasadded to the culture medium immediately upon transfec-tion and incubated for one day. Next, the cells were pas-saged and cultured in drug-free medium for three days. Fi-nally, Shld1 was added back again to re-stabilize the FKBP-YFP-PBase fusion for another three days. The typical im-ages from fluorescence microscopy at three turning pointswere shown in Figure 6B: (i) one day after treatment withShld1, (ii) three days after removing Shld1 and (iii) threedays after retreatment with Shld1. We also measured thepercentage of YFP positive cells every day by flow cytome-try. To control for plasmid dilution during the experiment,the percentages of YFP positive cells from the FKBP-YFP-PBase fusions were normalized by the percentage of YFPpositive cells in the corresponding control YFP-PBase sam-ples. The results are plotted in Figure 6C. Taking Figure 6Band C together, we observed a sharp drop in the YFP posi-tive cell population at the second measurement time point,indicating that without the inducer Shld1, the fusion pro-teins were quickly degraded. The normalized percentage ofYFP positive cells was nearly completely restored to theoriginal value three days after Shld1 was reapplied, indicat-ing the system is reversible. By fitting the curves in Figure6C to an exponential decay functions, we estimated the half-life of the FKBP-PB fusion protein to be 30 h. Our resultssuggested FKBP-based inducible PB transposon system istunable and reversible.


Figure 6. Tunability and reversibility of the FKBP-based PB transposon induction system. (A) FKBP-PBase-FKBP activity is tunable. Cells were trans-fected with donor and helper plasmids (FKBP-PBase-FKBP) and subjected to various concentrations of Shld1: 0 nM, 8 nM, 40 nM, 200 nM and 1"M. The normalized number of puromycin-resistant colonies observed is plotted versus the Shld1 concentration for four cell lines. (B) Shld-1 inductionis reversible. Fluorescent images are taken at various timepoints after induction with 1uM Shld1 or removal of Shld1 from transfected HEK293T cells.The white scale bar equals 50 "m. The red arrows indicate the time points of measurement. Bright field and fluorescent images were shown at top andbottom panel respectively. (C) Quantification of the reversibility of Shld-1 induction. The cells imaged in panel B were quantified by FACS. The normalizedpercentage of YFP positive cells is plotted at each time point for four cell lines.

Unlike 4OHT, Shld1 does not interfere with general cellularfunctions

It has previously been reported that 4OHT adversely af-fects developmental processes such as neurogenesis (23),myelinogenesis (23), myometrial differentiation (24) andsexual maturity (25). Therefore, we next sought to test ifcells treated with Shld1 or 4OHT display any developmen-tal phenotypes. We added these chemicals to EBs gener-

ated from mouse ESCs, and differentiated them into ven-tral spinal neural cells with retinoic acid and smoothenedagonist (28). We found that in the presence of 2 "M 4OHT,EB differentiation was inhibited and neuron-like cells werenot generated. In contrast, EBs that were mock-treated ortreated with 2 "M Shld1 differentiated normally (Figure7A). These results suggest that 4OHT inhibits EB differen-tiation, while Shld1 does not.


Figure 7. The effects of chemical inducer on RW4 mouse ES cells (ESCs). (A) Images of embryoid bodies (EBs) and differentiated EBs treated with (a)95% ethanol as vehicle, (b) 2 "M Shld1 and (c) 2 "M 4-hydroxytamoxifen during EB formation and neural differentiation from mouse ESCs to neurallineages. (B and C) The comparison of gene expression profile of drug-treated samples with that of mock-treated ones. Experiments were done in duplicate.Mean of the normalized counts from drug-treated samples was plotted against log2 fold change of drug-treated samples to mock-treated ones under thecategory of (B) Shld1 versus Mock and (C) 4OHT versus Mock. Red dots indicate the differentially expressed genes.

To further explore the effects of the inducer molecules ondifferentiation, we sought to quantify the extent to whichthese Shld1 and 4OHT perturb the transcriptional networkof EBs. EBs were treated with Shld1 and 4OHT, respec-tively, for 2 days, induced with retinoic acid and smoothenedagonist for 2 more days, and then subjected to gene expres-sion profiling by RNA-Seq. Cells mock-treated with 95%ethanol as vehicle were used as controls. For each condition,two biological replicates were performed and correlationco-efficiencies between them are above 0.96 (Supplemen-tary Figure S5), demonstrating reproducibility. The meanof the normalized counts from drug-treated samples wasplotted against log2 fold change of drug-treated samplesto mock-treated ones under the category of Shld1 versusMock and 4OHT versus Mock (Figure 7B and C). A sig-nificant change of gene expression profile was observed for4OHT-treated samples but not in Shld1-treated ones. Af-ter Benjamini–Hochberg correction for multiple hypothe-ses (see methods), we identified 260 differentially expressedgenes in the 4OHT-treated samples (Supplementary data 3).We performed gene ontology analysis on these genes usingthe DAVID Bioinformatics package (38,39) and functionalannotation clustering revealed that ‘heat shock’ and ‘stressresponse’ were the most over-represented terms (adjusted

P < 0.01), a result that supports our experimental obser-vation that 4OHT is toxic to EBs. In contrast, only onegene (FOS, NM 005252) was differentially expressed in theShld1 treated samples, suggesting that this chemical has lit-tle to no effect on the transcriptional network that controlsdevelopment of EBs.

DISCUSSION

We have systematically characterized, in four cell lines, 15different PB transposase fusion proteins representing threedifferent induction systems. We found that the FKBP-basedsystem achieved the broadest dynamic range of inductionacross four cell lines. Remarkably, in the presence of chem-ical inducer, this system had transposition efficiencies thatwere almost as high (∼95%) as the ‘wild-type’ PB transpo-son. Our results, coupled with the fact that Shld1 does notaffect ESC development, suggest that this will be the pre-ferred induction system for many types of experiments, es-pecially those involving cellular differentiation or organis-mal development.

In our plasmid-based experiments (Figure 3), we ob-served a lower dynamic range for the ERT2-based PB trans-poson induction system that was previously observed by


Cadinanos et al. (∼20-fold versus ∼270-fold) (9). This dis-crepancy is likely explained by the fact that, in our donorplasmid, the puromycin resistance gene is driven by a PGKpromoter whereas in Cadinanos et al., the puromycin re-sistance gene is promoterless and downstream of a splic-ing acceptor site. In that system, only insertions in activegenes will produced colonies, but in our system, all inser-tions will produce resistant clones. This would explain whywe observed more background puromycin-resistant cloneswhen the transposase was not induced since both studiesnormalized to background and small absolute changes inthe denominator can lead to large changes in the calculatedfold change.

The moderate background transposition of the DD in-duction system observed in our plasmid experiments (Fig-ure 3) was largely the result of the over-expressed fusion pro-tein overloading the cells’ proteasome system, since whenthe FKBP-fused PBase was placed under the control ofa cumate-titratable promoter and delivered at lower copynumber, we observed no background transposition whilestill achieving robust transposition after induction. Since nobackground hops were observed at several cumate concen-trations, the fold-induction of the system is not defined; weconservatively estimate it as >100-fold.

An interesting difference between DD-based systems andthe ERT2 based system is that the performance of theDD systems was more uniform across the difference celllines. We hypothesize that this difference is due to the factthat the DD system uses the ubiquitin–proteasome systemto constantly degrade FKBP-fused PBase. The ubiquitin–proteasome system is a general degradation machinery anduniformly expressed and works efficiently in all mammaliancell types. In contrast, the ERT2-based induction system isdependent on a specific cytoplasmic protein HSP90, a genewhose expression varies widely between cells. Our RNA se-quencing data showed that the HSP90 expression level inthe RW4 mouse ES cell line is about 4-fold higher than in theHCT116 cell line, suggesting that a low amount of HSP90levels may lead to either a high basal activity without induc-tion or a low induced activation.

In the FKBP-based PB transposon induction system, weobserved that N-terminally tagged PBase (FKBP-PBase)has a higher fold induction than the C-terminally taggedcounterpart (PBase-FKBP), which may suggest that FKBPfused at the N terminus is more likely to be exposed andthereby recognized by the proteasome than when fused tothe C terminus. This terminal specific higher activity may re-late to the distribution of functional components of PBase.It was reported that the PBase has a functional nuclear tar-geting signal in the 94 C-terminal residues (40). C-terminalfused PBase, therefore, is more likely to negatively affect thenuclear translocation of the PBase protein, which in turnincreases the likelihood of being recognized and degradedby the proteasome in the cytoplasm. We also observed thatfusing additional DDs to the PB transposase increases thefold-change in activation between the induced and unin-duced conditions. However, the addition of multiple DDscauses the absolute activity of the induced protein to besignificantly reduced, suggesting a trade off between howtightly a protein can be regulated, and its maximum activityin the induced state. Our results suggest that the FKBP-DD

is optimal when a high level of PB transposition is preferred,while FKBP-FKBP-PBase-FKBP is the choice when tightregulation is the priority.

The FKBP system will be likely prove considerably moreuseful than then ERT2 system for the study developmentalprocesses, since we found that the ERT2 inducer tamoxifenis a strong inhibitor of embryoid body differentiation. Ourresults are consistent with previous reports that tamoxifenhas deleterious effects on a number of developmental sys-tems (23–25). In contrast, we found that Shld1, the FKBPinducer, has no such phenotype, and furthermore, few tran-scriptional changes were observed in our RNA-Seq analysisof embryoid bodies treated with Shld1. Together, these re-sults suggest that the FKBP DD system is prefereable to theERT2 system for experiments involving cellular differentia-tion.

In summary, we have investigated the applicability ofthree different induction systems to the PB transposase bycharacterizing and optimizing 15 fusion constructs to de-velop a broadly applicable PB transposon induction sys-tem based on the FKBP degradation domain. This induc-tion system satisfies five important criteria: (i) The sys-tem has a low basal transposition activity when ‘off ’ andmaintains high transposition activity when ‘on’; (ii) ThePBase fusion protein shows high transposition activity al-most equal to that of the unfused ‘wild type’ PBase; (iii)The system can be applied across different cell lines withhigh performances; (iv) The induction is reversible andresponds in a dose-dependent manner; (v) The chemicalinducer does not interfere with general cellular function.Taken together, our experiments suggest that this systemcan be readily applied to diverse research applications suchas PB transposon-mediated genome engineering and tech-nology development.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENT

The RW4 Mouse ESC line was generously provided by DrDavid Gottlieb. The authors thank Dr Christian Shivelyand Dr Joe Dougherty for advice and useful discussions inpreparation of the manuscript. The authors are grateful toJessica Hoisington-Lopez and the Washington UniversityGenome Technology Access Center for their expert assis-tance with the use of Illumina Genome Analyzer. The au-thors thank the Alvin J. Siteman Cancer Center at Wash-ington University School of Medicine and Barnes-JewishHospital in St. Louis, MO, for the use of the Siteman FlowCytometry Core, which provided the Flow cytometry anal-ysis service. The Siteman Cancer Center is supported in partby an NCI Cancer Center Support Grant #P30 CA91842.

FUNDING

National Institutes of Health [U01MH109133 and5R01NS076993]; WM Keck Foundation Grant. Fundingfor open access charge: National Institutes of Health[U01MH109133 and 5R01NS076993].


Conflict of interest statement. None declared.

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